There is a strong desire for improved bioanalytical-sensor concepts compatible with detailed analysis of biorecognition events, including, for example, nucleotide-hybridisation, antibody-antigen recognition, drug-receptor interactions etc. In one common approach the analyte molecules (targets) to be recognized by immobilized receptor (probes) are labelled, e.g. with fluorescent or radioactive compounds. In alternative and increasingly important approaches, the biorecognition events are recorded without the introduction of external labels. The demand for label-free detection originates primarily from the observations that: (i) molecules to be detected from complex mixtures are complicated to label 25 in a rapid, reproducible and homogeneous manner, (ii) labels may interfere with the actual biorecognition event and (iii) information from binding kinetics can generally not be achieved, which thus complicates affinity and concentration determinations. Significant progress in this direction has recently been made, thus allowing label-free and sensitive detection of various biorecognition events. Among such analytical methods are optical methods such as SPS/SPR surface plasmon spectroscopy/resonance), (Rich and Myszka 2000) ellipsometry and OWLS (optical waveguide light spectroscopy), (Ramsden 1993) piezoelectric methods such as QCM (quartz crystal microbalance) or SAW (surface acoustic wave) (Janshoff and others 2000) and fluorescent methods such as SPFS (surface plasmon induced fluorescence spectroscopy) (Liebermann and Knoll 2000) and fluorescence imaging (Niemeyer and Blohm 1999). Out of these, SPR is the far most widespread technique (Rich and Myszka 2000. Except for a novel optical design allowing highly sensitive detection of changes in the refractive index at the interface between a gold surface and a liquid, generally an aqueous solution, the technology is compatible with microfluidics for handling of small sample volumes and imaging of patterned surfaces. (Jordan and others 1997) In addition, a variety of gold-surface-modification protocols designed for efficient immobilization of various types of biomolecules have been successfully developed.
However, while the protocols developed for immobilization of water soluble proteins, such as antibodies and many enzymes, as well as oligonucleotides have been proven efficient and reliable, membrane proteins have been shown more cumbersome to handle. This is indeed a severe complication, since supported cell membrane mimics on solid supports aids the fundamental functional studies of e.g. photosynthesis, respiration and neurobiology.
Furthermore, since membrane proteins, especially transmembrane proteins, constitute an important class of proteins, this challenging problem is critical also with respect to pharmaceutical applications, not the least since the majority of drugs are directed towards membrane proteins. The fundamental complication in proper handling of membrane proteins originates from the fact that they, in contrast to water-soluble proteins, carry hydrophobic membrane segments, which must be shielded from water in order for the protein to sustain in its native conformation. This shielding can either be achieved by the use of detergents, which keep the protein soluble in aqueous solution, or preferably by reconstitution of protein into cell-membrane mimicking structures, such as, for example, liposomes or planar supported bilayers. This, in turn, puts strong requirements on the immobilization strategies. In order to develop strategies compatible with immobilization of lipid bilayer assemblies on solid supports, including incorporated membrane proteins, several strategies have been developed. The most straight forward one utilizes spontaneous adsorption, decomposition and fusion of intact vesicles into planar supported bilayers on Si02, glass or mica-surfaces (Brian and McConnell 1984; Burgess and others 1998; Gizeli and others 1997; Granèli and others 2003; Gritsch and others 1998; Heyse and others 1998; Kalb and Tamm 1992; Lindholm-Sethson 1998; Salafsky and others 1996. However, since the water-soluble parts of membrane proteins incorporated in planar supported bilayers have a tendency to interact directly with the solid support, this strategy has been shown to have a negative influence on the mobility and activity of the protein (Salafsky and others 1996). In addition, the bare presence of the protein may in certain cases interfere with the actual bilayer formation process. (Granèli and others 2003) One promising way to circumvent the former problem is to use a spacer or cushion, often an inert soft polymer, between the protein and the solid support, (Naumann and others 2002; Wagner and Tamm 2000) and EP 07847939; or to create membranes that span small cavities on the surface (Schmidt and others 2000. However, in situations when direct electrical access to both sides of the membrane is 35 not a prerequisite, the use of immobilized intact vesicles may avoid the problems related to the influence from the solid support on the function of the membrane protein, (Cooper and others 2000; Svedhem and others 2003), or the influence from the membrane proteins on the actual bilayer formation process. (Granèli and others 2003) It has been demonstrated how vesicles can be immobilized on a transducer surface utilizing (i) spontaneous binding to a solid support (e.g., Au, Ti02, Pt) (Keller and Kasemo 1998; Reimhult and others 2002), (ii) a fraction of lipids in the vesicles designed to bind specifically to one type of functional entities on a surface (e.g. vesicles containing biotin-modified lipids coupled to streptavidin coated surfaces (Jung and others 2000; Michel and others 2002) or antibody-antigen based coupling (MacKenzie, 1997), (iii) hydrophobic tags immobilized on the transducer surface, (Cooper and others 2000) or (iv) DNA-modified vesicles for specific coupling to DNA modified surfaces, (Patolsky and others 2000) also compatible with array formats. (Svedhem and others 2003)
Furthermore, in comparison with planar supported lipidbilayers, the use of immobilized vesicles enhances the potential number of target sites (e.g. membrane proteins) that can be immobilized per surface area, even in comparison with strategies in which detergent depletion under controlled flow conditions are used to increase the concentration of immobilized membrane proteins in planar supported lipid bilayers. (Karlsson and Lofas 2002; Karlsson and Löf{dot over (a)}s 2002) However, it is generally difficult to incorporate membrane proteins with large hydrophilic domains at high concentration in liposomes since the protein then tend to aggregate and lose in activity (see e.g. (Richard and others 1990 and references therein).
Hence, even in situations when immobilized vesicles are used, the surface concentration of proteins must often be kept relatively low. It is therefore of outmost importance to develop strategies where the amount of immobilized membrane protein is increased without significantly influence their function.